Margaret J. Gordon

Preferential uptake of long chain polyunsaturated fatty acids by isolated human placental membranes

Fatty acid uptake by the placenta is thought to be a carrier-mediated process, however the mechanism by which long chain polyunsaturated fatty acids (LCPUFA) are preferentially accumulated from the maternal circulation to the fetal tissues is still unclear. To examine the role of the placenta in this process, binding of four different radiolabelled fatty acids ([14C]oleate, [14C]linoleate, [14C]a-linolenate and [14C]arachidonate) to human placental membranes was studied. Binding of fatty acid was found to be time- and temperature dependent. At equilibrium, the total binding of oleate was highest (5.1 ± 0.1 nmoles/mg protein) followed by linoleate (2.8 ± 0.31 nmoles/mg protein) and arachidonate (2.06 ± 0.4 nmoles/mg protein) and α-linolenate binding was lowest (0.5+0.1 nmoles/mg protein). However, oleate had the lowest specific binding (37% of the total binding) whereas arachidonate had the highest specific binding (∼ 86% of the total binding) followed by linoleate and a-linolenate (62%, and 69% of the total binding, respectively). Binding of each [14C] fatty acid was also assessed in the presence of 20-fold excess of other unlabelled ligands. Binding sites seem to have preference for the binding of [14C] fatty acids in the following order: arachidonic acid ⋙ linoleic acid ≫ a-linolenic acid ⋙≫ oleic acid, whereas BSP and a-tocopherol did not show any competition with any of the [14C] fatty acids. These data suggest that the fatty acid binding sites in placental membranes are specific for the fatty acids but that they have heterogeneous affinities.Trans fatty acids (elaidic and linoelaidic acids) also competed very strongly for the [14C] fatty acid binding. Polyclonal antiserum raised against placental FABPpm inhibited binding of these [14C]fatty acids but with variable degrees of inhibition; EFA/LCPUFA binding was much more than that of oleate. Our data suggest that EFA/LCPUFA bound to albumin are preferentially transported by human placental membranes and that the placental FABPpm may be involved in the sequestration of EFA/LCPUFA by the placenta.

Placental membrane fatty acid-binding protein preferentially binds arachidonic and docosahexaenoic acids.

To elucidate further the role of placental membrane fatty acid-binding protein (p-FABPpm) in preferential transfer of maternal plasma long chain polyunsaturated fatty acids (LCPUFA) across the human placenta, direct binding of the purified protein with various radiolabelled fatty acids (docosahexaenoic, arachidonic, linoleic and oleic acids) was investigated. Binding of these fatty acids to the protein revealed that p-FABPpm had higher affinities and binding capacities for arachidonic and docosahexaenoic acids compared with linoleic and oleic acids. The apparent binding capacities (Bmax) values for oleic, linoleic, arachidonic and docosahexaenoic acids were 2.0 +/- 0.14, 2.1 +/- 0.17, 3.5 +/- 0.11, 4.0 +/- 0.10 mol per mol of p-FABPpm whereas the apparent dissociation constant (Kd) values were 1.0 +/- .0.07, 0.73 +/- 0.04, 0.45 +/- 0.03 and 0.4 +/- 0.02 microM, respectively (n=3). In the case of human serum albumin, the Kd and Bmax values for all fatty acids were around 1 microM and 5 mol/mol of protein, respectively. These data provide direct evidence for the role of p-FABPpm in preferential sequestration of maternal arachidonic and docosahexaenoic acids by the placenta for transport to the fetus by virtue of its preferential binding of these fatty acids.

Effects of tomato extract on human platelet aggregation in vitro

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 w l of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 2 g supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

Vitamin E requirements, transport, and metabolism : role of α-tocopherol-binding proteins

Abstract Vitamin E (RRR-α-tocopherol) is a lipid-soluble antioxidant that is present in the membranes of intracellular organelles. There it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E that differ in their structure and in biological activity in vivo and in vitro. Although γ-tocopherol is a more effective free radical scavenger than α-tocopherol in vitro, the reverse is true in vivo, suggesting that the tocopherol distribution systems favor the localization of α-tocopherol at the sites where it is required. Vitamin E is transported in plasma primarily by lipoproteins, but little is known of how it is transported intracellularly. A 30 kDa α-tocopherol-binding protein in the liver cytoplasm may regulate plasma vitamin E concentrations by preferentially incorporating the vitamin E homologue, RRR-α-tocopherol (α-tocopherol), into nascent very low density lipoproteins. However, this α-tocopherol-binding protein is unique to the hepatocyte, whereas α-tocopherol is present in the cells of all major tissues. Moreover α-tocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required; in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. This raises the question of how the lipid-soluble α-tocopherol is transported intracellularly in different tissues. We have identified a new α-tocopherol-binding protein of molecular mass 14.2 kDa in the cytosol of heart and liver. This protein specifically binds α-tocopherol in preference to the δ- and γ-homologues but does not bind oleate. Studies on immunoreactivity and ligand specificity of the protein suggest that it is not a fatty acid-binding protein. The 14.2 kDa α-tocopherol-binding protein stimulates the transfer of α-tocopherol from liposomes to mitochondria in vitro by 8 to 10 fold. We suggest that this low molecular mass TBP may be responsible for the intracellular transport and distribution of α-tocopherol in the tissues.

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.

DIFFERENTIAL DISTRIBUTION AND METABOLISM OF ARACHIDONIC ACID AND DOCOSAHEXAENOIC ACID BY HUMAN PLACENTAL CHORIOCARCINOMA (BEWO) CELLS

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.

Plasma membrane fatty acid-binding protein (FABPpm) of the sheep placenta

Fatty acid-binding protein (FABPpm) has been identified and characterised from sheep placental membranes. Binding of [14C]oleate to placental membranes was found to be time- and temperature-dependent. Addition of a 20-fold excess unlabelled oleic, palmitic, or linoleic acid reduced the binding of [14C]oleate to the membranes to around 50% of total binding, whereas D-alpha-tocopherol at similar concentrations did not affect [14C]oleate binding. This indicates that the binding sites are specific to fatty acids. Specific binding of [14C]oleate was reduced by heat denaturation or trypsin digestion of the membranes, suggesting that the fatty acid-binding sites are protein in nature. FABPpm was then solubilised from sheep placental membranes, and subsequently purified to electrophoretic homogeneity using an oleate-agarose affinity column. The purified FABPpm had an apparent molecular mass of 40 kDa, as determined by SDS-PAGE and by gel permeation chromatography. The [14C]oleate-binding activity of the purified protein was also confirmed by PAGE followed by autoradioblotting. The specific binding for oleate was around 1.5 nmol per mg of membrane protein. Our data indicate the presence of FABPpm in sheep placental membranes.

Purification and partial characterisation of an α-tocopherol-binding protein from rabbit heart cytosol

An α-tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 μg per g of tissue. The binding of α-tocopherol to the purified protein was rapid, reversible, and saturable. Neither γ nor δ tocopherol could displace the bound α-tocopherol from the protein, suggesting a high specificity for α-tocopherol. α-Tocopherol-binding protein did not bind oleate. Transfer of α-tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of α-tocopherol in the heart.

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [(14)C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A(2) synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.

Acyl-CoA thioesterase activity in human placental choriocarcinoma (BeWo), cells: effects of fatty acids

The effects of fatty acids on acyl-CoA thioesterase activity and peroxisome proliferator-activated receptor gamma (PPARgamma), a regulator of lipid metabolism, were investigated in placental choriocarcinoma (BeWo) cells. Substrate preference for acyl-CoA thioesterase was in the following order; gamma-linolenoyol-CoA>/=arachidonoyol-CoAz.Gt;palmitoyl-CoA>/=linoleyol-CoA. However, when these cells were incubated with fatty acids, acyl-CoA thioesterase activity was increased by both conjugated linoleic and gamma linolenic acids, but not by docosahexaenoic and eicosapentaenoic acids. In addition, these fatty acids also increased expression of PPARgamma in these cells, suggesting a putative relationship between free fatty acid generated by acyl-CoA thioesterase and expression of PPARgamma. Since expression of PPARgamma is critical for feto-placental growth, these fatty acids may be important during pregnancy.